PROJECT SUMMARY The malaria parasite Plasmodium has to accomplish its sporogonic development in the mosquito vector in order for malaria transmission to occur. The long-term goal of the PI's research is to elucidate the interactions between the mosquito vector and malaria parasite to identify transmission-blocking targets suitable for the development of disease control strategies. During its journey through the mosquito, the parasite engages in intimate interactions with the mosquito midgut, hemolymph, and salivary gland and relies on numerous mosquito-derived host factors (agonists). Plasmodium can be impaired in the vector by agonist deletion through gene editing (GM mosquito population replacement), depletion through RNA interference (RNAi-based control through GM mosquito population replacement or external application of dsRNAs or analogues), or blocking/interference/inhibition through antibodies (transmission blocking vaccines) or small molecules (transmission blocking drugs or mosquito exposure). Hence, an obvious advantage of studying Plasmodium agonists for malaria control is that they can be targeted through multiple means. The recently developed CRISPR/CAS9-based gene editing in Anopheles mosquitoes provide new and promising opportunities for the study of agonist function through gene deletion, and for malaria control through gene drive. Here we will focus on 10 pro-Plasmodium factors (agonists) to assess their potential for malaria control and further our knowledge of their involvement in Plasmodium transmission. In Aim 1, we will validate 4 putative Plasmodium agonists (that have previously only been tested with non-human parasites) for P. falciparum transmission-blocking potential in A. gambiae using RNAi-mediated gene silencing. In Aim 2, we will develop CRISPR/CAS9- mediated gene KO mosquitoes for selected P. falciparum agonists. In Aim 3, we will assess these KO A. gambiae lines for resistance to P. falciparum infection and their fitness impact in terms of mosquito longevity and fecundity.